Confocal microscopy involves scanning a tissue to produce microscopic images of a slice or section of tissue. Such microscopic imaged sections may be made in-vivo and can image at cellular resolutions. Examples of confocal scanning microscopes are found in Milind Rajadhyaksha et al., “In vivo Confocal Scanning Laser Microscopy of Human Skin: Melanin provides strong contrast,” The Journal of Investigative Dermatology, Volume 104, No. 6, June 1995, pages 1-7, and more recently, in Milind Rajadhyaksha et al., “Confocal laser microscope images tissue in vivo,” Laser Focus World, February 1997, pages 119-127. These systems have confocal optics which direct light to tissue and image the returned reflected light. Such confocal microscope systems can focus and resolve a narrow width of tissue as an imaged section, such that tissue structures can be viewed at particular depths within the tissue, thereby avoiding evasive biopsy procedures for pathological examination of the tissue, or allow pathological examination of unprepared excised tissue.
Two parameters which effect the performance of confocal microscope systems in imaging tissue sections are the numerical aperture (NA) of the optics and the wavelength of the beam scanned through the tissue. The axial resolution, i.e., the thickness of the imaged section, and lateral resolution of confocal microscope systems are directly proportional to the wavelength of the light source and inversely proportional to NA2 (axial) and NA (lateral). In other words, the higher the NA, the thinner the imaged section, while the lower the NA, the thicker the imaged section. Both the axial resolution and the lateral resolution are optimized in a confocal microscope system suitable for pathological examination to the dimensions of the tissue structures, such as cells, which are of interest. As discussed in the Milind Rajadhyaksha et al. article appearing in Laser Focus World, February 1997, the use of a near-infrared light source between about 700 nm and 1200 nm and optics with a NA of about 0.7-0.9 have provided optimal results for imaging tissue sections with sufficient discrimination of cellular level structures. One problem with using optics providing NA values about this range is that they are large and expensive, particularly for the objective lens which focuses light into and collects light from the tissue, and are very sensitive to aberrations, such as introduced by the object being imaged. Accordingly, it is desirable to provide imaging of tissue sections in a confocal microscope using lower cost and smaller optics having a NA below 0.7 without sacrificing imaging performance, in particular depth discrimination and scattered light rejection.
Accordingly, it is a feature of the present invention to improve confocal microscopy by combining the depth response of confocal imaging with the coherence function of heterodyne detection using a synthesized beam of multiple wavelengths of light, such that lower NA confocal optics and inexpensive laser diode sources may be used. Heterodyne detection has been proposed for imaging in U.S. Pat. No. 5,459,570, which describes an apparatus using an optical coherence domain reflectometer for providing images of a tissue sample to perform optical measurements. However, this apparatus is limited in depth resolution and does not utilize confocal optics for microscopic imaging. Other optical systems have used multiple wavelengths of light, but are limited to generating interference patterns for visualizing fringes characterizing the surface of objects, such as shown in U.S. Pat. No. 5,452,088, which describes a multi-mode laser apparatus for eliminating background interference, and U.S. Pat. No. 4,632,554, which describes a multiple frequency laser interference microscope for viewing refractive index variations. Such interferometric-based optical systems have no confocal optics or heterodyne detection, nor do they provide imaging within a tissue sample. A confocal microscope using multiple wavelengths of light has been proposed in U.S. Pat. No. 4,965,441, but this microscope is limited to focusing at different altitudes for surface examination of an object and does not have heterodyne detection.